A novel bioluminescent protease assay using engineered firefly luciferase

Abstract

Proteases play important roles in a variety of disease processes. Understanding their biological functions underpins the efforts of drug discovery. We have developed a bioluminescent protease assay using a circularly permuted form of firefly luciferase, wherein the native enzyme termini were joined by a peptide containing a protease site of interest. Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold. The mutant luciferase substrates are easily generated by molecular cloning and cell-free translation reactions and thus the protease substrates do not need to be chemically synthesized or purchased. The assay has broad applicability using a variety of proteases and their cognate sites and can sensitively detect protease activity. In this report we further demonstrate its utility for the evaluation of protease recognition sequence specificity and subsequent establishment of an optimized assay for the identification and characterization of protease inhibitors using high throughput screening.

Fig. (1). Schematic Representation of the CP234-Luc Assay.

Firefly luciferase is a 61 kDa monomeric enzyme that catalyzes the oxidation of firefly luciferin in the presence of ATP and oxygen to emit yellow-green light. Upon binding of substrates, the structure of firefly luciferase undergoes conformational changes from open to closed forms. We created a circularly permuted luciferase by covalently joining the native N and C termini of firefly luciferase through the cloning in of a short polypeptide linker containing a protease recognition sequence. This results in restricting the movement between the two domains and locking the enzyme in the less active open form. Protease cleavage releases this constriction thereby restoring higher activity. A. To express this mutant luciferase, new N and C termini were inserted at amino acids 234 and 233, respectively. B. Insertion of the polypeptide linker greatly reduces luciferase activity. Proteolytic cleavage by the cognate protease (scissors) activates the mutant luciferase enzyme resulting in a luminescent signal in the presence of the luciferin substrate (yellow circle).

Fig. (2). Evaluation of Sites for Circular Permutation.

A. Cleavage dependent activation of CP-Luc/ENLYFQS fusion protein. Cell-free translation reactions were diluted 1:1 in 2X TEV protease buffer, incubated at 30°C for 30 minutes ± 10 U TEV protease and luminescence was measured from 5 µL aliquots. Data are the mean values, N = 3. Error bars are the standard deviation of the mean. B. Five µL of the protease digest reaction was size-fractionated on NuPAGE^® gels and FluoroTect^TM labeled proteins were visualized on a fluoroimager. TEV protease digestion resulted in the expected sized fragments. Note, the digestions were not complete, and therefore, residual undigested 60 kDa protein remains in all of the “+” lanes. No expressed protein is visible in the no DNA lanes.

Fig. (3). Evaluation of TEV Protease Recognition Sequence P₁' Specificity.

Cleavage dependent activation of CP₂₃₄-Luc/ENLYFQX fusion proteins. Cell-free translation reactions were diluted 1:1 in 2X TEV protease buffer, incubated at 30°C for 30 minutes ± 10 U TEV protease and luminescence was measured from 5 µL aliquots. The X-axis is the amino acid at the P₁' position (X) of the CP₂₃₄-Luc/ENLYFQX fusion protein. The Y-axis is the fold activation of the CP₂₃₄-Luc/ENLYFQX fusion proteins calculated by dividing the mean TEV digested values by the mean undigested values (no TEV digest), N = 3. Error bars are the standard deviation of the mean.

Fig. (4). TEV Protease Titration.

TEV protease dose dependent activation of CP₂₃₄-Luc/ENLYFQS protein. Cell-free translation reactions were diluted 1:1 in 2X TEV protease buffer, incubated at 30°C for 30 minutes with titrating amounts of TEV protease and luminescence was measured from 5 µL aliquots in 100 µL 1:1 diluted Bright Glo^TM Luciferase Assay reagent in dH₂O; N = 4. Results are plotted as signal to noise (S/N). The limit of detection was defined as the amount of TEV protease giving a S/N ratio = 3 (dotted line). The R² value was 0.99.

Fig. (5). Representative Data from Plate 1 of the LOPAC¹²⁸⁰ Library Screen.

The LOPAC¹²⁸⁰ library was screened for TEV protease inhibitors using the CP₂₃₄-Luc/ENLYFQC assay. Shown here are the data from plate 1 (N = 320 compounds). There were three hits () based on greater than 60% TEV protease inhibition, blue line (= 345,311 RLU). The CP₂₃₄-Luc/42AA assay was also performed to detect potential non-specific affectors of the CP₂₃₄-Luc assay and the results, which are overlaid on the graph in gray, indicate that two compounds non-specifically affected the assay. () = CP₂₃₄-Luc/42AA activity in DMSO; () = CP₂₃₄-Luc/42AA activity with compound library; () = CP₂₃₄-Luc/ENLYFQC activity minus TEV enzyme in DMSO; () = CP₂₃₄-Luc/ENLYFQC activity plus TEV enzyme in DMSO, and () = CP₂₃₄-Luc/ENLYFQC activity plus TEV protease with compound library. Controls are plotted as the mean of N = 64 (CP₂₃₄-Luc/42AA in DMSO) or 32 (CP₂₃₄-Luc/ENLYFQC ± TEV protease in DMSO). Error bars are the standard deviation of the mean.

Fig. (6). Inhibitor Potency Titration Curves.

Five compounds were re-tested at titrating concentrations. The five compounds were: aurintricarboxylic acid (ATA), 8,8'-[carbonylbis(imino-3,1-phenylenecarbonylimino)]bis(1,3,5-naphthalene-trisulfonic acid) hexasodium salt (NF-023), 4-[3-(4-Acetyl-3-hydroxy-2-propylphenoxy)propoxy]phenoxyacetic acid (L-165,041), SCH-202676 hydrobromide or N-(2,3-diphenyl-1,2,4-thiadiazol-5-(2H)-ylidene)methanamine hydrobromide (SCH HBr), and 6-hydroxyl-DL-DOPA or 2,5-dihydroxy-DL-tyrosine (L-DOPA). A. Using the CP₂₃₄-Luc/ENLYFQC assay, NF-023 and ATA were tested from 0 – 0.2 mM and the other three compounds were tested from 0 – 1 mM. Data are the mean values, N = 3. Error bars are the standard deviation of the mean. B. Using the protein fusion cleavage assay, compounds were tested from 0 – 0.1 mM. Data are the average of N = 2.

Fig. (7). Chemical Structures of Three TEV Inhibitor Compounds.

Three compounds found in the LOPAC¹²⁸⁰ library screen as TEV inhibitors were confirmed using both the CP₂₃₄-Luc/ENLYFQC assay and the protein fusion cleavage assay. Their chemical structures are: A. aurintricarboxylic acid (ATA), B. 8,8'-[carbonylbis(imino-3,1-phenylenecarbonylimino)]bis(1,3,5-naphthalene-trisulfonic acid) hexasodium salt (NF-023), C. 4-[3-(4-Acetyl-3-hydroxy-2-propylphenoxy)propoxy]phenoxyacetic acid (L-165,041).